Science of the Total Environment 512–513 (2015) 540–551

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Future water quality monitoring — Adapting tools to deal with mixturesof pollutants in water resource management

Rolf Altenburger a,b,⁎, Selim Ait-Aissa c, Philipp Antczak d, Thomas Backhaus e, Damià Barceló f,Thomas-Benjamin Seiler b, Francois Brion c, Wibke Busch a, Kevin Chipman g, Miren López de Alda f,Gisela de Aragão Umbuzeiro h, Beate I. Escher i,a, Francesco Falciani d, Michael Faust j, Andreas Focks k,Klara Hilscherova l, Juliane Hollender m, Henner Hollert b, Felix Jäger n, Annika Jahnke a, Andreas Kortenkamp o,Martin Krauss a, Gregory F. Lemkine p, John Munthe q, Steffen Neumann r, Emma L. Schymanski m,Mark Scrimshaw o, Helmut Segner s, Jaroslav Slobodnik t, Foppe Smedes l, Subramaniam Kughathas o,Ivana Teodorovic u, Andrew J. Tindall p, Knut Erik Tollefsen v, Karl-Heinz Walz w, Tim D. Williams g,Paul J. Van den Brink k, Jos van Gils x, Branislav Vrana l, Xiaowei Zhang y, Werner Brack a

a UFZ — Helmholtz Centre for Environmental Research, Permoserstr. 15, 04318 Leipzig, Germanyb RWTH Aachen University, Aachen, Germanyc Institut National de l'Environnement Industriel et des Risques INERIS, BP2, 60550 Verneuil-en-Halatte, Franced Centre for Computational Biology and Modelling, University of Liverpool, L69 7ZB, UKe Department of Biological and Environmental Sciences, University of Gothenburg, Carl Skottbergs Gata 22b, 40530 Gothenburg, Swedenf Water and Soil Quality Research Group, Institute of Environmental Assessment and Water Research (IDAEA-CSIC), Jordi Girona 18-26, 08034 Barcelona, Spaing School of Biosciences, The University of Birmingham, Birmingham B15 2TT, UKh University of Campinas, Limeira, Brazili National Research Centre for Environmental Toxicology (Entox), The University of Queensland, Brisbane, Australiaj Faust & Backhaus Environmental Consulting, Fahrenheitstr. 1, 28359 Bremen, Germanyk Alterra, Wageningen University and Research Centre, P.O. Box 47, 6700 AA Wageningen, The Netherlandsl Masaryk University, Research Centre for Toxic Compounds in the Environment (RECETOX), Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republicm Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf, Switzerlandn Synchem UG & Co. KG, Am Kies 2, 34587 Felsberg-Altenburg, Germanyo Brunel University, Institute of Environment, Health and Societies, Uxbridge UB8 3PH, United Kingdomp WatchFrog, Bâtiment Genavenir 3, 1 rue Pierre Fontaine, 91000 Evry, Franceq IVL Swedish Environmental Research Institute, P.O. Box 53021, 400 14 Göteborg, Swedenr Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle, Germanys University of Bern, Centre for Fish and Wildlife Health, PO Box 8466, CH-3001 Bern, Switzerlandt Environmental Institute, Okruzna 784/42, 97241 Kos, Slovak Republicu University of Novi Sad, Faculty of Sciences¸ Trg Dositeja Obradovića, 321000 Novi Sad, Serbiav Norwegian Institute for Water Research NIVA, Gaustadalléen 21, N-0349 Oslo, Norwayw MAXX Mess- und Probenahmetechnik GmbH, Hechinger Straße 41, D-72414 Rangendingen, Germanyx Foundation Deltares, Potbus 177, 277 MH Delft, The Netherlandsy State Key Laboratory of Pollution Control & Resource Reuse, School of the Environment, Collaborative Innovation Center for Regional Environmental Quality, Nanjing University, Nanjing 210023,PR China

Abbreviations:AA, annualaverage;AOP, adverseoutcomepathways;BQE,biological qualityelements;CIS,CommonEuropean implementationstrategy;DGSANCO,DirectorateGeneralforHealth andConsumerProtection of the EuropeanCommission;EDA, effect-directed analysis; EQS, environmental quality standards; EROD, ethoxyresorufin-O-deethylase; EU, EuropeanUnion;GC–MS/MS, gas chromatographycoupledwithdoublemass spectrometry;GFP, greenfluorescentprotein;GST, glutathionesulfotransferases;HPCCC,highperformance counter cur-rent chromatography; KE, key event; LC–HRMS/MS, liquid chromatography of high resolution coupledwith doublemass spectrometry;MAC,maximumallowed concentrations;MIE,mo-lecular initiating event;MoA,mode of action; PAH, polycyclic aromatic hydrocarbons; PNEC, predicted no-effect concentration; RBSPs, river basin specific pollutants; TU, toxic units;WFD,Water FrameworkDirective.⁎ Corresponding author at: UFZ — Helmholtz Centre for Environmental Research, Permoserstr. 15, 04318 Leipzig, Germany.

http://dx.doi.org/10.1016/j.scitotenv.2014.12.0570048-9697/© 2015 Elsevier B.V. All rights reserved.

H I G H L I G H T S G R A P H I C A L A B S T R A C T

541R. Altenburger et al. / Science of the Total Environment 512–513 (2015) 540–551

• Future water contamination monitoringcan address the detection of priority

mixtures.

• Effect-based tools will help to assess theimpact of mixture on water quality.

• Drivers of mixture toxicity can be iden-tified using effect-directed analysis.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 15 October 2014Received in revised form 18 December 2014Accepted 18 December 2014Available online 31 January 2015

Editor: E. Capri

Keywords:WFDWater qualityPriority chemicalsChemical statusEffect-based toolsEcological statusMixture toxicity

Environmental quality monitoring of water resources is challenged with providing the basis for safeguarding theenvironment against adverse biological effects of anthropogenic chemical contamination from diffuse and pointsources. While current regulatory efforts focus on monitoring and assessing a few legacy chemicals, many moreanthropogenic chemicals can be detected simultaneously in our aquatic resources. However, exposure to chem-ical mixtures does not necessarily translate into adverse biological effects nor clearly shows whether mitigationmeasures are needed. Thus, the question which mixtures are present and which have associated combined ef-fects becomes central for defining adequate monitoring and assessment strategies. Here we describe the visionof the international, EU-funded project SOLUTIONS, where three routes are explored to link the occurrence ofchemical mixtures at specific sites to the assessment of adverse biological combination effects. First of all,multi-residue target and non-target screening techniques covering a broader range of anticipated chemicalsco-occurring in the environment are being developed. By improving sensitivity and detection limits for knownbioactive compounds of concern, new analytical chemistry data for multiple components can be obtained andused to characterise priority mixtures. This information on chemical occurrence will be used to predict mixturetoxicity and to derive combined effect estimates suitable for advancing environmental quality standards. Second-ly, bioanalytical tools will be explored to provide aggregate bioactivity measures integrating all components thatproduce common (adverse) outcomes even for mixtures of varying compositions. The ambition is to providecomprehensive arrays of effect-based tools and trait-based field observations that link multiple chemical expo-sures to various environmental protection goals more directly and to provide improved in situ observations forimpact assessment of mixtures. Thirdly, effect-directed analysis (EDA) will be applied to identify major driversof mixture toxicity. Refinements of EDA include the use of statistical approaches with monitoring informationfor guidance of experimental EDA studies. These three approaches will be explored using case studies at theDanube and Rhine river basins as well as rivers of the Iberian Peninsula. The synthesis of findings will beorganised to provide guidance for future solution-oriented environmentalmonitoring and exploremore system-atic ways to assess mixture exposures and combination effects in future water quality monitoring.

© 2015 Elsevier B.V. All rights reserved.

1. Introduction

The monitoring of freshwaters with the goal of safeguarding envi-ronmental water quality in Europe so far has focused on the evaluationof the ecological and chemical status of water bodies. For the ecologicalstatus biological and hydromorphological quality elements are consid-ered, while the chemical status is judged based on consideration of afew selected compounds (EU Dir, 2000/60; EU Dir, 2013/39). Theestablished techniques for the biological quality elements rely onphytoplankton, macrophytes, phytobenthos, benthic invertebrate,and fish fauna recordings (EU Dir, 2000/60). These monitoring ef-forts are carried out on a wide scale and at regular intervals, suchthat the ecological status is the aggregate of occurrence and abun-dance information. The chemical status, on the other hand, is derivedfrom information on analytically determined concentrations of pri-ority pollutants in different compartments such as water, sediment

and biota, which are compared against Environmental Quality Standards(EQS) (EU Dir, 2008/105; CIS GD 27, 2011). Complementary efforts in-clude emission monitoring, effluent testing for acute toxic effects, andrisk management measures for specific products, such as buffer zonesfor pesticide application or product labelling for pharmaceuticals or con-sumer products.

Despite the enormous efforts, the picture that emerges regardingecological and chemical status is still incomplete, fragmented, andwith contradictory assessments of the situation. There is general con-sensus that the target of “good ecological status” defined in the WaterFramework Directive (WFD) will not be reached for the majority ofEuropean water bodies within the anticipated timeframes (EEA,2012). Among the causes for this failure the contribution of chemicalcontamination, however, remains unclear, although efforts to assesschemical monitoring results point to a contributory role of chemicalcontamination (Malaj et al., 2014). Overall, about 40% of European

Fig. 1. Challenges to deal with mixtures of pollutants in water quality monitoring and toprovide management solutions.

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water bodies (EC COM 673, 2012) still have an unknown chemicalstatus as not even the monitoring of the EU-wide priority substanceshas been performed. From a management perspective the legacy com-pounds are of diminishing importance, due to decreasing use of thesesubstances (many are regulated or banned) and the growing awarenessthat many other chemicals occur and may cause adverse effects in theaquatic environment. The occurrence of anthropogenic chemicals inthe environment appears indeed to be widespread and the detectionof mixtures of contaminants seems to be the rule rather than the excep-tion (Kolpin et al., 2002; Loos et al., 2009). While elaborated hazard as-sessments leading to environmental quality standards are performedfor priority pollutants, this is not the case for most other chemicalsthat have been recently detected. This is why these may be referred toas contaminants of emerging concern (EPA, http://water.epa.gov/scitech/cec/).

The European Commission became aware of the problem ofchemical mixtures (Council conclusions, 2009), and in its communi-cation on the combination effects of chemicals (EC COM 252, 2012)describes the challenges requiring scientific support. In principle,tools for analysing and assessing combined effects from definedmix-tures have been well studied and documented over the past decades(e.g., Kortenkamp and Altenburger, 2011) and suggestions abouthow component-based predictive environmental risk assessmentmay be performed are presented (e.g., Backhaus and Faust, 2012).Thus, the existence of combined effects is a fact and the principalmeans of addressing them are known (EC, 2011). The challenge now

Table 1Environmental Quality Standard (EQS), annual average (AA) and maximum allowable concensurface watersa (EU Dir., 2013/39/EU). Unit: μg/l, nomenclature as in the legal reference.

AA-EQSInland surface waters

Dicofol 1.3 × 10−3

Perfluorooctane sulfonic acid and its derivatives (PFOS) 6.5 × 10−4

Quinoxyfen 0.15Aclonifen 0.12Bifenox 0.012Cybutryne (Irgarol) 0.0025Cypermethrin 8 × 10−5

Dichlorvos 6 × 10−4

Hexabromocyclododecane (HBCDD) 0.0016Heptachlor and heptachlor epoxide 2 × 10−7

Terbutryn 0.065

a Inland surface waters encompass rivers and lakes and related artificial or heavily modified

is to develop systematic ways of addressing chemical mixtures in envi-ronmental assessment (EC COM 252, 2012).

The EU-fundedSOLUTIONSproject (http://www.solutions-project.eu/)takes up this challenge for water quality assessment and monitoringby undertaking to improve monitoring strategies and combiningthem with modelling efforts based on pre-market data (Brack et al.,2015). Here we outline our strategies for analysing and assessingchemical mixtures for water quality monitoring purposes. We intendto explore three options for identifying and developing systematicapproaches to accommodate for contaminant mixtures in waterquality assessment (Fig. 1). Firstly, we test the hypothesis that it ispossible to identify mixtures whose compositions are representativefor specific sites or typical for specific sources and are thus amenableto component-based mixture assessment. Secondly, we elaboratemeans of identifying batteries of bioanalytical assays that allow com-prehensive assessment of impact of mixtures on water quality. Final-ly, we combine effect-based and chemical analytical tools to probecausal links between mixture occurrence and combined effects andto support the identification of drivers of mixture toxicity.

The major questions of combination effects of chemicals (EC COM252, 2012) with regard to their impact on water quality assessmentand the abovementioned strategies will be studied in the context ofcase studies at the river Danube (de Deckere et al., 2012; Grundet al., 2011; Liska et al., 2008), the Rhine catchment (Hollenderet al., 2009; Ter Laak et al., 2010) and for rivers of the Iberian Peninsula(Muñoz et al., 2009; Navarro-Ortega et al., 2012). Investigations willbe based on existing data and experimental studies. Moreover, thesecase studies will be utilised to complement and jointly evaluate re-sults from modelling and measurement-based approaches (Bracket al., 2015).

2. Identification of priority mixtures

The Scientific Committees of the Directorate General for Health andConsumer Protection (DG SANCO) have emphasised that ‘in view of thealmost infinite number of possible combinations of chemicals […] focuson mixtures of potential concern is necessary’ (EC, 2011). A number ofcriteria were proposed for consideration, including co-occurrence at in-dividual concentrations belowbut close to acceptable levels, indicationsfor similar action, and the potential for toxicological interactions.Additional criteria, such as scale of exposure (EC COM 252, 2012), co-occurrence of transformation products or source attributions might beconsidered. In general, if bias towards known contaminants is to bereduced, this task requires on the one hand multi-residue target andnon-target screening techniques to cover mixtures occurring in the en-vironment more comprehensively. On the other hand, improvementsleading to lower detection limits for known bioactive compounds arealso needed as for some of the newly established water priority sub-stances (Table 1) it is currently virtually impossible to analytically

trations (MAC) set for the newly established WFD priority substances in inland and other

AA-EQSOther surface waters

MAC-EQSInland surface waters

MAC-EQSOther surface waters

3.2 × 10−5 – –

1.3 × 10−4 36 7.20.015 2.7 0.540.012 0.12 0.0120.0012 0.04 0.0040.0025 0.016 0.0168 × 10−6 6 × 10−4 6 × 10−5

6 × 10−5 7 × 10−4 7 × 10−5

0.0008 0.5 0.051 × 10−8 3 × 10−4 3 × 10−5

0.0065 0.34 0.034

water bodies.

Table 2Chemical analytical problems addressed in the SOLUTIONS project to support priority mixture identification.

Problem Approach Method Aim References

Compound detection below EQSand estimation of time-averagedconcentrations

Enrichment of trace compounds bytime-integrative passive sampling

Partitioning and adsorptionbased passive sampling; Flowcontrolled passive sampling

Widen applicability of passivesampling by extending the methoddomain on emerging compounds andimprove their performance in termsof limits of quantification andmeasurement uncertainty

Lohmann et al.(2012), Smedes andBooij (2012), Vrana(2012), Vermeirssenet al. (2013), Moschetet al. (2014b)

Time-integrated sampling by in sitularge volume solid phase extraction

Large-volume sampler forapplication in situ (e.g., at pointsources or on monitoring ships)

Development of routinely applicableand commercially available techniquewith negligiblecompound-dependence of extractionefficiency; applicable for chemicaland biotesting in parallel

Schulze et al. (2014)

Hydrodynamic counter currentchromatography (HPCCC)

HPCCC–liquid–liquidpartitioning

Improved enrichment and clean up asmethod improvements for wider use

Ignatova et al. (2011)

On-line extraction and clean upmethodology for LC

Turbulent flow chromatography Automated on-line enrichmenttechnique and clean up

Lopez-Serna et al.(2012)

Inadequate coverage ofenvironmental mixturecomponents

Automated workflows for routinelyapplicable target and non-targetscreening techniques

GC– and LC–HR MS/MStechniques with innovativesoftware tools and parameterprediction

Detection, identification andsemi-quantification of largernumbers of chemicals at the sametime including unknowns

Schriks et al. (2010),Vadillo and Barceló(2012), Schymanskiet al. (2014), Krausset al. (2010)

Structure elucidation procedures forenvironmental trace contaminantsand transformation products byintegration of analyticalinformation from GC– andLC–HRMS/MS with prediction toolsfor retention, MS fragmentation,hydrogen–deuterium exchange andmass spectral and compounddatabases

Workflow integrating analyticaltechniques and the use ofdatabases and software in aconsensus lines of evidenceapproach

Identification of new chemicalsincluding transformation productsand other unknowns in variousmatrices

Zonja et al. (2014),Huntscha et al.(2014), Schymanskiet al. (2014), Gerlichand Neumann (2013),Hug et al. (2014)

Total contaminant concentrationsin sediment do not reflect theexposure, i.e., biologicallyaccessible concentration, becauseof unknown uptake capacity ofsediments

Availability-based approach for theassessment of sedimentcontamination using equilibriumpartitioning passive sampling; bothnon-depletive (chemical activity)and depletive (accessible)

A release isotherm is recordedby equilibrations at differentsampler — sediment ratiosproviding both the level in porewater and the accessibleconcentration.

Obtaining measured concentrationsfrom sediment samples that allowspatial comparison and conversioninto units applicable in other matrices(water, lipid) for comparisonbetween environmentalcompartments.

Reichenberg andMayer (2006),Smedes et al. (2013)

Detection and unravelling ofinternal contamination of biotawith trace contaminants

In tissue passive sampling to assessinternal exposure to environmentalmixture

Silicone thin-films as‘chemometers’ equilibrated inintact tissues

Measure of the complex mixturespresent in tissue while leaving thematrix behind

Jahnke et al. (2009,2014)

Parallel detection of multiplecontaminants and selectedbiomarkers

LC–MS/MS screeningapproaches for contaminantsand marker proteins

Integrated assessment ofcontamination and biochemicalresponse

Yang et al. (2015)

Improved sample clean-up fordetermination of biotaconcentrations

Selective extraction andclean-up for lipid removal

Solving matrix problems for thedetection of a broad set of emergingpollutants

Huerta et al. (2013),Navarro-Ortega et al.(2012)

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determine compounds at the very low EQS concentrations set for themin the WFD.

It is therefore the goal of the SOLUTIONS project to improve chemicalanalytics both with respect to capabilities to screen for more com-pounds and to improve present detection limits. Subsequently, thedata from case studies will be utilised to investigate the co-occurrenceof components. To identify mixtures of priority, two data evaluationstrategies will be pursued. Firstly, we will try to identify patterns ofco-occurring compounds and correlate them to site characteristics,land use or specific contamination sources. Secondly, to support the as-sessment of detected mixtures, toxicity data gaps will be filled throughmodelling and subsequent hazard quotient formulation. The results willbe used in component-basedmixture toxicity extrapolations to identifymixtures of potential toxicological concern (Price and Han, 2011).

The significant analytical gaps regarding the detection limits of com-pounds with very low PNECs or EQS (Table 1) in environmental mediaand/or biota require novel concepts in the sampling and clean-up ofsamples. With a given sensitivity of chemical analytical techniques,detection limits can be improved by accumulating and concentratingcompounds from larger volumes of water, e.g., either by passive sam-pling or by large volume solid phase extraction. Table 2 lists the

approaches that are pursued to this end and summarises the existingexperience within the SOLUTIONS consortium.

The number of analytical methods developed for targeted determi-nation of emerging contaminants has experienced rapid growth overrecent years and continues to increase which has led to the discoveryof new environmental contaminants, metabolites and transformationproducts. Major gaps remain with respect to the identification andelucidation of the structure of known and unknown components ofcomplex environmentalmixtures potentially composed of tens of thou-sands of components. Two recent studies (Malaj et al., 2014; Moschetet al., 2014a) demonstrated that more comprehensive analytical com-pound screening may substantially alter the assessment of surfacewater quality. In the study of Moschet et al. (2014a), five Swiss riverinecatchments were sampled during spring and analysed for the occur-rence of some 250 components, mainly pesticides and biocides. AA-EQS exceedances for 19 compounds occurred in 70% of the water sam-ples. This observation would have escaped attention when restrictingthe assessment to priority components only. Malaj et al. (2014) provideevidence that compounds occurring in European freshwaters even forroutinely monitored chemicals such as γ-hexachlorocyclohexane, atra-zine, cyanide, chlorpyrifos, chlorfenvinfos, or diuron at their detected

Fig. 2. Heatmap of concentrations for 141 chemicals reported in Moschet et al. (2014a) in five rivers, clustered to identify occurring mixture patterns. MDL= minimum detection limit.

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concentrationsmay be close to hazardous concentrations at many sites.A second finding was that the outcome of risk assessment criticallydepends on the number of compounds analysed: often, apparentlylow environmental risk associates with a limited number of monitoredchemicals. After these proof-of-principle investigations, subsequentsteps should therefore address the question of how to assess the totalityof hazardous contamination in a reliable way while at the same timekeeping efforts at a realistic level. To address this issue a focus on prior-itymixtures thatmight be derived from chemical analytical informationis a promising approach. Prioritymixture identification based on the an-alytical data is, in our perspective, not limited to sets of definedchemicals at specified concentrations but rather an analysis of patternsis needed as described above.

SOLUTIONS looks for answers regarding better coverage of detect-able and unidentified compounds by establishing non-target screeningworkflows and a set of interacting compound identification toolswhich integrate GC–MS/MS and LC–HRMS/MS technology with com-puter tools for retention, fragmentation, hydrogen–deuterium ex-change and toxicity prediction and database for mass spectra. Moredetails concerning the roads taken are summarised in Table 2.

Oncewe obtainmore comprehensive data on the occurrence ofmul-tiple chemicals in freshwaters by means of targeted, multi-residue, andscreening chemical analytical efforts, the subsequent issue will be tofind outwhethermixture patterns can be elucidated. In order to identifypotentially repetitive mixture patterns, analytical data for detectedcompounds could be subjected to data clustering. An exemplary effortis illustrated in Fig. 2.

Here, out of 396 organic compounds that were analysed and quanti-fied in water samples from five small rivers of the Rhine catchment(Moschet et al., 2014a), 141 chemicals were found to occur abovetheir detection limits in at least one of the rivers. The data was hier-archically clustered (distance method = “Euclidian”, clusteringmethod = “Ward”) according to the site of occurrence and the detect-ed concentrations. At this coarse level, groups of chemicals with high,moderate and low concentrations can be determined and site-specificoccurrences become obvious. Using this approach for comparing moresites including additional chemical, toxicological (e.g., hazard ratios),or site-specific information may be advanced to allow characteristictoxicological signatures to be correlatedwith the different human activ-ities such as the cultivation of grains, orchards or meadows as opposed

Fig. 3. Conceptual framework for bioanalytical tools illustrating their place in an adverse outcombioanalytical tools in mixture impact assessment.

to urban, domestic, or industrial influences. Moreover, the scale ofoccurrence of mixtures and archetypical versus river basin-specific pol-lutants may be derived.

Efforts such as those from Malaj et al. (2014) and Moschet et al.(2014a) not only provide wider coverage of priority pollutants and cur-rently used pesticides than previously available, but also demonstratethat the detectable concentrations may raise concern for unwanted bio-logical effects. To study the significance, temporal and spatial scale of oc-curring concentrations, complementary comparison with toxicityinformation for the detected compounds should help. Subsequently,any concentration–response-relationship information can feed intocomponent-based mixture toxicity modelling approaches (Altenburgeret al., 2004; Altenburger andGreco, 2009) to derive estimates of resultingcombined effect. The results of these combined effect estimates may inturn prove to be suitable for the development of a novel perspective foridentification of river basin-specific pollutants and for advanced EQS set-tings for priority mixtures.

3. Impact of mixtures

Chemical monitoring of water quality accounts for quantitative as-sessments of the occurrence and fate of known contaminants in waterbodies and thus facilitates themanagement and remediation of definedcompounds. The ultimate goal of water quality management under theWFD, however, lies in the provision of good ecological and chemicalstatus. Thus, analytically undetected but toxicologically relevantcompounds, transformation products and mixture effects may beoverlooked in an approach that is purely based on chemical analyticalmeasurements. It is suggested that bioanalytical tools can improve theenvironmental impact assessments (CMEP, 2014; Escher and Leusch.,2012; Malaj et al., 2014). A second goal in SOLUTIONS, therefore, is toadvance and apply bioanalytical methods to see whether improved im-pact assessment ofmixtures iswithin reach. The simultaneous exposureof organisms to different compounds may not necessarily mean thatcombined effects are evoked at detectable levels (Altenburger et al.,2004). This may be due to individual components acting differentlyand itmay be due to the relation between the dose-dependency of com-ponents and the concentrations found in the mixtures which may notgive rise to detectable contributions (EC, 2011). A way forward for mix-ture impact assessment for field situations may be seen in devising

e pathway network elucidated by mixture exposure and indicating the potential roles of

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bioanalytical tools that are tailored for specific mixture assessmentobjectives.

Bioanalytical tools are defined here as assays which capture keyevents (KE) of biological reactions following experimentally controlledor observed chemical exposure and molecular initiating events (MIE)in an organism, detected at the level of the cell, organism, populationor community and possibly leading to adverse outcomes. Moreover,these tools can inform us about the existing toxic pressure for biologicalsystems if employed in situ. The first large scale attempts have recentlybeen made to address the use of various bioassays for mixture impactanalysis of surfacewaters (Carvalho et al., 2014, Escher et al. 2014). Sub-sequently demonstrating that effects of mixtures seem to be relevant invarious environmental settings, differentmanagement perspectives canbe distinguished. The management problem may need (i) diagnostics,i.e., identifying the biological receptor that is affected by mixture expo-sure; (ii) forensics, i.e., elucidating the causes of an emerging adverseeffect and their responsible source; or (iii) status assessments,i.e., allocating the contribution of chemicals to an impaired ecologicalstatus and delivering a prognosis for the development of the waterquality.

The underlying conceptual thinking in the SOLUTIONS project forbenchmarking the studied bioanalytical tools with respect to their con-tributions for the different mixture impact questions will be based on amodified version of the concept of adverse outcome pathways (AOPs)(Ankley et al., 2010; OECD, 2013) as illustrated in Fig. 3. In distinctionto the AOP concept we here deal with mixtures, where it is conceivedthat no longer individual molecular initiating events but rather mea-sures of common adverse outcome are required to capture potentialmixture impacts (EFSA, 2013). We thus define key events as those

Table 3Bioanalytical tools used in the SOLUTIONS project to improve the impact assessment of mixtur

Biological level Biosystem Response observation In

Key events Feral fish EROD activity, bile PAH metabolites InGST activity In

Mammalian andFish cells

EROD activity DiNuclear receptor activation/inhibition Oe

Mammalian andYeast cells

Nuclear receptor activation/inhibition An

Mammalian cells Fish nuclear receptor inhibition Co

Isolated enzyme Acetylcholine–esterase activity NeCellular responses E. coli, yeast Gene expression, alterations on

proliferation of geneStr

Salmonellatyphimurium

Ames test using diagnostic strains M

Mammalian cellline

p53 activation Ge

Nrf2 protein in AREc32 activation Ox

NF-kappaB activation InFish cells Immune gene modulation Im

Organism responses Zebrafishembryo

Oestrogenic cyp 19a1b-GFP activation Oe

Medaka embryo Oestrogenic choriogenin-GFP activation OeAndrogenic spiggin-GFP activation An

Xenopus embryo Thyroid THbZIP-gfp activation ThAlgae Growth, transcriptome ApDaphnids Motility, transcriptome, metabolome Ap

Zebrafishembryo

Development, transcriptome Ap

Thyroid disruption EnAbramis abramis Histopathology Or

Community responses Algal biofilms Community tolerance measured as14C-uptake and biofilm formationkinetics

Ec

Invertebrates Alterations of trait composition Ec

EDA — effect-directed analysis.MOA — mode-of-action.

observations that integrate several potential MIEs. This would comprisenot only simultaneous observation of activation or inhibition of variousnuclear receptors but also detecting alterations of biotransformationwhich under mixture exposure can provide indication for unexpectedcombined effects. In the AOP at the next level of biological complexitycellular stress responses and subsequently organism fitness measuresare observed.

Effect-based tools summarise all the various cell- or organism-basedbioassays that typically are performed in the lab to characterise environ-mental samples. Effect-based tools with response detection on themolecular, subcellular or cellular levels are believed to aggregate thecombined effects of similar bioactive components for the specific re-sponses they are designed to capture. For diagnostic or forensic tasks ar-rays of tools will have to be designed to cover different biological effectqualities, while for surveillance tasks where a defined receptor is to beprotected, individual tools might provide effective impact detectors.

Effect-based tools that detect apical organism responses are easilyrelated to toxicologically consented adverse effects and thus lend them-selves to applications in chemical environmental hazard assessment.Mixture impact assessment is currently well capable of assessing thecombined toxicity of similar and dissimilar acting components at the or-ganism level (Altenburger and Greco, 2009), whereas understandingthe translation of mixture responses observed in molecular and cellularassays andmore apical and regulatory-relevant assays remains a formi-dable research challenge (Altenburger et al., 2012). Therefore, bylinking the responses from the different organisational levels throughthe integrated use of bioassays representing the molecular, cellular, or-ganism and population levels we aim to improve our understanding ofpotential biases in the existing effect detection tools.

es for diagnostic, forensic and ecological quality purposes.

dication of Project aim Method reference

ternal exposure In situ exposure Brinkmann et al. (2013)ternal exposure – Kammann et al. (2014)oxin-like EDA detector Creusot et al. (2013a)strogen/anti-oestrogen – Creusot et al. (2013b)drogen/anti-androgen – Jalova et al. (2013)

rticosteroid/anti-corticoid – Kugathas and Sumpter(2011)

urotoxicity EDA detector Holth and Tollefsen (2012)ess-response activation EDA detector Zhang et al. (2011), Su

et al. (2014)utagenicity EDA detector Umbuzeiro et al. (2011),

Reifferscheid et al. (2012)notoxicity Adaptive stress

responseKnight et al. (2009), Yehet al. (2014)

idative stress – Wang et al. (2006), Escheret al. (2012)

flammation as immune response – Knight et al. (2009)mune-competence – Segner et al. (2012)strogen/anti-oestrogen Validation of

cellular responseindication; EDAdetector

Brion et al. (2012), Fetteret al. (2014)

strogen/anti-oestrogen – Kurauchi et al. (2005)drogen/anti-androgen – Sébillot et al. (2014)yroid/anti-thyroid EDA detector Fini et al. (2007)ical effects, MOA Effect diagnostics Nestler et al. (2012)ical effects, MOA – Meland et al. (2011),

Williams et al. (2011)ical effects, MOA – Büttner et al. (2012),

Klüver et al. (2011)docrine activity – Schmidt et al. (2012)gan toxicity In situ effects Wolf et al. (2010)ological mode-of-action In situ effects Blanck (2002), Pesce et al.

(2010)

ological mode-of-action – Van den Brink et al. (2011)

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Finally, ecological tools are employed to bridge toxicological effectfindings as understood for individual organisms and chemical mixturesfrom the effect-based tools, to field observations of compromised eco-logical structure and function. Two perspectives are pursued here, onthe one hand for selected effects, such as exposure stimulated metabo-lism we perform in situ studies on feral fish (Brinkmann et al., 2013;Boettcher et al., 2010) (Table 3) while on the other handwewill deploytrait-based approaches to investigate community-level effects ofchemical contaminants. Trait-based approaches are used increasinglyto derive correlations between the occurrence of species traits andexposure to (mixtures of) chemicals, but also to distinguish be-tween chemical stress effects and impact of other major pressures,e.g., hydromorphological alterations or eutrophication. If mode of ac-tion (MoA)-specific species traits can be identified, biomonitoringdata could be used as a marker for chemical stress at the aggregatingMoA level. This assessment can also be used to identify the chemicalslikely to pose the highest ecological risks (Van den Brink et al., 2013).

A variety of bioanalytical tools will be explored in this project(Table 3) for their capabilities to aggregate mixture effects of chemicalsirrespective of the presence of possibly unknown chemicals, or variabil-ity in the mixture composition. The list is not comprehensive butcomprises (i) in vitro nuclear- and cell-reporter assays that indicate in-tracellular presence of contaminants or detect specific receptor- or ag-gregated stress responses, (ii) standard toxicological organism-basedbioassays that detect apical responses in fish, daphnia and algae anddirectly relate to established biological quality elements (BQE), and(iii) ecology-oriented bioindicators using biomarker responses in indi-viduals or community function (pollution-induced community toler-ance), or trait-based composition information. The bioanalytical toolsto be applied in the SOLUTIONS project are further specified inTable 3 regarding their properties, perspective and the existingexperience.

Bioanalytical tools in their totality and in future arrays could thushelp determine the impact of mixtures with respect to distinct water

Fig. 4. Principles of a tiered effe

quality management questions. Moreover, if proven workable, thisapproach could possibly link multiple chemical exposure assessmentdirectly to specific environmental protection goals.

4. Identification of mixture toxicity drivers

Despite the presence ofmixtures ofmultiple compounds in environ-mental media and samples, theoretical considerations and experimen-tal findings suggest that the overall risk may be driven by only a fewmixture components (Altenburger et al., 2004; Backhaus and Karlsson,2014; Price et al., 2012). The European Commission considers the devel-opment of methodologies for the identification of such drivers of mix-ture toxicity a research priority (EC COM 252, 2012). One of the majorchallenges in the assessment of complex environmentalmixtures there-fore is the identification of those chemicals that contribute significantlyto observed effects. Furthermore, routinely detected chemicals oftencannot explain observed biological responses (e.g., Escher et al., 2013)which points to a mismatch between these assessment approaches.This mismatch may be resolved through joint efforts from both disci-plines for the different lines of evidence, e.g., by linking chemical mon-itoring and biological effect and monitoring data by traits-based oreffect-directed approaches.

Effect-directed analysis (EDA) may help to identify novel and unex-pected compounds that may cause adverse effects on biota and humanhealth (Brack et al., 2008). The principle of EDA is to reducenatural sam-ples to less complex mixtures or individual compounds by bioassay-directed fractionation of environmental samples so that relevanttoxicants can be isolated and identified. The approach has been demon-strated as useful in several instances (Brack, 2011; Houtman et al., 2007;Thomas et al., 2009) and will be advanced and applied on water,sediments and fish from selected sites in the river basins of Danube,Rhine, and beyond. Current limitations of EDA due to laborious andtime-consuming procedures will be addressed by SOLUTIONS. This in-cludes specific investigations on the application of EDA for monitoring,

ct-directed analysis (EDA).

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structure elucidation of unknown polar compounds, increasing thenumber of bioanalytical endpoints, and the application to food chainaccumulation and thus secondary poisoning.

The approach pursued is illustrated in Fig. 4. SOLUTIONS will devel-op a tiered protocol to identify river basin-specific pollutants that can beconsidered drivers of mixture toxicity. To date, the monitoring ofcontaminants according to WFD is restricted to chemical analyticalmonitoring of individual chemicals. In the first tier this informationcan be used for the establishment of MoA that are known to be relevantin specific water bodies or river basins supporting a MoA- or BQE-specific default approach e.g., based on the summation of toxic unitsof the components (Backhaus and Faust, 2012). This approach alreadygoes beyond the current WFD approach and provides a first set ofchemical target screening-based candidate drivers. The MoA infor-mation also helps to complement chemical monitoring with multi-endpoint (eco)toxicological screening and allows for the identificationof mismatches between candidate drivers and multiple biological ef-fects. If unexplained biological effects occur, WFD-like chemical targetmonitoring is extended in tier 2 bymulti-target and non-target screen-ing in order to achieve a more comprehensive picture of contaminationpatterns. In combination with (eco)toxicological screening, this pro-vides the basis for a novel approach called virtual EDA to identify chem-ical signals that are correlated with effects from background signals.Virtual EDA has been suggested as a term by Eide et al. (2002) andhas been recently evaluated in a proof of concept study for the charac-terisation of chemicals responsible for mutagenic effects in a river im-pacted by an industrial effluent (Hug et al., in prep.). The approachreduces the complexity of mixture components through the use ofmultivariate statistics and pattern recognition methods on samples forseveral sites as a virtual decomposing approach which should directthe focus of subsequent more elaborated identification efforts to asubset of sites. SOLUTIONS will test this approach in case studies oncontaminated samples from theDanube and Rhine river basins. Still un-explained mixture effects will be addressed through higher tier EDAstudies (tier 3) as a site-specific approach, which will also be used tovalidate the results of virtual EDA at specific sites.

The identification of unknown compounds usingmass spectrometrydata remains a major bottleneck in many disciplines (Creek et al., 2014;Scheubert et al., 2013) and often hinders the successful completion ofEDA studies (Schymanski et al., 2009). Efforts in SOLUTIONS will there-fore focus on the development of methods for generating and pre-selecting toxicant candidate structures from the given analytical andeffect information as indicated in Table 2. The structure elucidationapproaches also include efforts for the integration of prediction of trans-formation, toxicity, physico-chemical properties, MS fragmentation andchromatographic retention.

The diagnostic power of higher tier EDA will be addressed throughefforts to adapt assays for specific key events (see Table 3) as effectdetectors for EDA. An array of screening assays potentially coveringmultiple species, MoAs and adverse effects (see Table 3 for details)will be deployed in the EDA approach.

Moreover, food chain accumulation will be approached exem-plarily for fish tissue to investigate bioaccumulation and secondarypoisoning through feed and food contaminated with complex mix-tures of pollutants. Performing EDA on such tissue will aim to detectand identify bioavailable and bioaccumulative toxicants (Houtmanet al., 2004), including metabolites formed in the organisms (Jeonet al., 2013).

5. Perspectives for solution-oriented mixture assessment

A central deliverable of the SOLUTIONS project is to generateguidance for the three mixture assessment challenges identified by theEuropean Commission (EC COM 252, 2012), namely (i) the characterisa-tion of priority mixtures, (ii) mixture impact assessment, and (iii) theidentification of toxicity drivers. The need to tailor environmental

monitoring tools towards contamination diagnosis in complex environ-mental matrices, however, is acknowledged on a worldwide scale. Forexample, Environment Canada (2014) suggests guidance to use effect-based methods for aquatic effects monitoring from pulp and paper pro-duction. In Australia, where the water cycle is an issue with the perspec-tive of reuse for humans, strict standards for a larger number of potentialhazardous compounds have been formulated and it is suggested to linkchemical and bioanalytical tools for water quality monitoring (Tanget al., 2014).

Thus the goals set out here should be of a wider interest. To achievethem we will provide documentation of the chemical analytical andbioanalytical tools and specify the approaches for the different needsin water quality monitoring and assessment. The various problems incurrent water quality management call for tailored approaches, whichcould provide solution-oriented mixture assessments. For instance,the identification of river basin-specific priority groups of pollutants(RBSPs) needs to be improved for river basin management plans,while risk assessment for unwanted effects calls for a more prominentrole of bioanalytical tools. Mixture assessment is essential for waterquality management, given the complexity of typical pollution scenari-os. The tools that will be provided by the SOLUTIONS project shall facil-itate achieving this aim. The task is to operationalise the requiredmixture assessment, i.e., to tailor the available tools for the specifictasks laid out above. The SOLUTIONS project as a whole sets out to notonly provide advanced methodologies for water quality monitoring,but also to deliver suggestions for testing requirements and dataneeds for carrying out mixture risk assessment and management inthe context of theWFD. The last step will be performed in collaborationwith the modelling, case studies and conceptual framework activities(Brack et al., 2015).

The NORMAN network (http://www.norman-network.net/) hasrecently proposed a novel risk assessment-based approach forprioritisation of water pollutants for improving water monitoring(Dulio and von der Ohe, 2013; Brack et al., 2012). It suggests a strategyto cope with scarce data for individual compounds and to account fordifferentmanagement action categories. The scheme, however, remainslimited to individual compound assessments. The tools developed andthe data generated within the SOLUTIONS case studies may be used toamend such prioritisation schemes to address mixtures of contami-nants of emerging concern and their impacts explicitly.

The larger vision of future water resource management and thecontributions that can be anticipated, bears yet another level of perspec-tives. It iswidely acknowledged that Europeanwater bodies are affectedby multiple types of stress, such as water scarcity, morphologicalchanges, and pollution. Addressing the joint effects from such mul-tiple stressors in management is limited by the currently availableknowledge (Hering et al., 2014; Navarro-Ortega et al., 2014). Twointernational EU-funded projects, MARS (Hering et al., 2014) andGLOBAQUA (Navarro-Ortega et al., 2014), are addressing severalprimary and secondary stressors such as water flow extremes, ther-mal extremes, eutrophication, and impaired habitat morphology.The efforts in SOLUTIONS are clearly complementary and issuesare easily identified where joint efforts could improve our mecha-nistic understanding of interactions between say low water flowand the impact of pollution. Also, as risk assessment, WFD statusassessment and the understanding of ecosystem services followdifferent but related frameworks (Hering et al., 2014), we couldgain improved coherence by providing better understanding ofeach of the frameworks. Finally, we could learn to consistentlyaddress scaling issues from the water body through the river basinup to the continental scale.

The revision of the WFD in 2019, the ongoing discussion on a com-mon European implementation strategy (CIS), as well as the cycle ofreadjustments and refinements of river basin management planning(RBMPs) will be the outreach targets for our research activities. Timelyprovision of validated chemical analytical or bioanalytical tools,

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improved knowledge and useful decision support instruments will bevital for translating the various ideas into better practises. Moreover,an improved understanding of how mixture assessment may beperformed could generate incentives for more coherent approachesin water resource management by providing the means for cross-compliance measures in environmental regulation.

Acknowledgments

The SOLUTIONS Project is supported by the Seventh FrameworkProgramme (FP7-ENV-2013) of the European Union under grant agree-ment no. 603437.

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